1. Field of the Invention
The present invention relates to an E. coli producing and secreting human granulocyte-colony stimulating factor(hG-CSF), more specifically, to a recombinant plasmid constructed to express secretory hG-CSF in E. coli, an E. coli transformed with the said plasmid to secrete hG-CSF, and a process for preparing hG-CSF using the said transformed E. coli. 
2. Description of the Prior Art
Colony stimulating factors(CSFs) known to be synthesized by various cell types such as mononuclear macrophages, T-lymphocytes, and fibroblasts are found in the various parts of normal human body. CSFs are classified into three major categories, granulocyte-colony stimulating factor(G-CSF), macrophage-colony stimulating factor(M-CSF) and granulocyte/macrophage-colony stimulating factor(GM-CSF), among them, G-CSF is an essential protein in manufacturing blood cells via promoting proliferation and differentiation of hemopoietic stem cells, and facilitates increase in numbers of granulocytes, especially, neutrophils which play an important role in the protection of the body from the infection. Chemotherapies widely used to treat growing tumors not only inhibit the growth of tumors but also inhibit the production of neutrophils, giving rise to severe side effects due to the diminished protecting function of neutrophils. Administration of G-CSF to the patients under such chemotherapies is known to be an effective way of treatment and prevention of the infectious diseases by means of facilitating the increase in neurophil numbers.
In 1986, the hG-CSF gene was isolated from a human squamous carcinoma cell line CHU-II, its nucleotide sequence was first determined and expressed in COS cells by Nagata et al. (see: Nagata et al., Nature 319: 415 (1986)). The hG-CSF is a glycoprotein comprising 30 signal peptides which consists of 174 amino acid residues. The hG-CSF includes 5 cysteine residues of which 4 cysteines form two disulfide bonds, between Cys-36 and Cys-64, and between Cys-64 and Cys-74, which serve for folding of the expressed protein and its activity (see: Hill et al., Proc. Natl. Acad. Sci., USA, 90:5167–5171(1993)). The hG-CSF dose not have the consensus sequence(Asn-X-Thr/Ser) for N-glycosylation, but O-glycosylation occurs at Thr-133. However, the recombinant G-CSF produced in E. coli is known to have almost the same biological activities as natural G-CSF, which means glycosylation is unnecessary for the G-CSF activity.
With the recent progress in recombinant DNA technology, G-CSF can be produced in bacteria, plant cells and animal cells, and some results previously reported are described here: Souza et al. isolated a cDNA from the human bladder cancer cell line 5637, determined its sequence and reported its expression in E. coli (see: Souza et al., Science, 232:61(1986)). Moreover, researches on production of hG-CSF in E. coli, plant cells and animal cells, and on construction and production of hG-CSF derivatives have been reported. However, technologies known to date for the production of hG-CSF in E. coli have many disadvantages in terms of protein yield or production cost since the hG-CSF is produced in cytoplasm in the form of insoluble inclusion body, which requires subsequent solubilization and renaturation to obtain biologically active form of hG-CSF protein. Although small quantity of soluble hG-CSF can be isolated directly, such method still have limitations in a sense that the active fraction of hG-CSF protein has to be isolated from the pool of enormous amounts of E. coli proteins.
In general, proteins secreted to the periplasm of E. coli carry signal sequence, which is found in all proteins transportable out of the cytoplasm, and cleaved off by signal peptidase in the periplasm. The signal sequence is essential in secreting proteins in E. coli. Therefore, recombinant proteins originally not encoded by E. coli genes can be secreted into the periplasm or to the extracellular broth by joining known signal sequence(OmpA, OmpF, PelB, PhoA, SpA, etc.), as it is or with slight modifications, to the N-terminus of gene coding an exogenous protein.
The method for production of hG-CSF by secretion into the periplasmic space has following advantages over the conventional method by producing in cytoplasm described above: first, it is easy to isolate and purify the recombinant proteins, with high purity, in periplasm than in cytoplasm, since there are fewer proteins in periplasm than in cytoplasm (see: Nossal, N. G. et al., J. Biol. Chem., 241:3055–3062(1966)); secondly, recombinant proteins secreted into periplasm are segregated from the cytoplasm where the most proteases are found, obtaining high yield in production of recombinant protein by avoiding degradation of the protein by proteases present in cytoplasm (see: Meerman and Georgiou, Ann. N.Y. Acad. Sci., 721:292–302(1994)); thirdly, the bacterial periplasm is more oxidizing environment than cytoplasm, conducting disulfide bond formation and correct folding of polypeptide easily, thus, the formation of insoluble aggregates is avoidable (see: Hockney, TIBTECH, 12:456–463(1994)).
Having such advantages, the method resulting in secretion of recombinant proteins into the periplasm has been employed for production of hG-CSF in E. coli and reported as follows: Perez-Perez et al. have tried to get secreted form of hG-CSF, employing OmpA which is one of the signal sequences in E. coli, without success. To solve that problem, they employed the system for coexpression of two molecular chaperones, DnaK and DnaJ, and merely obtain a small quantity of secreted hG-CSF (see: Perez-Perez et al., Biochem. Biophys. Res. Commun., 210:524–529(1995)); and, Chung et al. have tried to obtain secreted form of hG-CSF employing another signal sequence, PelB, again without success, but hG-CSF was accumulated in the form of insoluble inclusion body in cytoplasm (see: Chung et al., J. Fermen. Bioengin., 85:443–446(1998)).
In view of above situation, there is a continuing need to develop the technique for facilitating secretion of hG-CSF at a substantial level into the periplasm through rather simple process which does not require solubilization of insoluble inclusion body and subsequent refolding.